1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
A variety of chemical routes to 1,3-propanediol are known. For example, 1,3-propanediol may be prepared from ethylene oxide and a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; by the catalytic solution phase hydration of acrolein followed by reduction; or from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and hydrogen over periodic table group VIII catalysts. Although it is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants.
Biological routes to 1,3-propanediol are known which utilize feedstocks produced from renewable resources. For example, bacterial strains able to convert glycerol into 1,3-propanediol are found e.g., in the species Klebsiella, Citrobacter, Clostridium, and Lactobacillus. In these bacteria, glycerol can enter either an oxidative or reductive pathway. Oxidation of glycerol results in the conversion of glycerol to dihydroxyacetone (DHA) by glycerol dehydrogenase and the DHA is phosphorylated by an adenosine triphosphate (ATP) dependent kinase to yield dihydroxyacetone phosphate (DHAP) which enters the glycolytic pathway in the cell. Reduction of glycerol is accomplished by an initial isomerization and dehydration catalyzed by glycerol dehydrates to yield 3-hydroxypropionaldehyde which is further reduced by 1,3-propanediol:NAD.sup.+ oxidoreductase to yield 1,3-propanediol, a dead end cellular metabolite. The expression of at least the first two enzymes involved in the oxidative pathway as well as the two enzymes involved in the reductive pathway in K. pneumoniae are coordinately regulated. The four enzyme system is functionally linked where the production of 1,3-propanediol from glycerol is dependent on the presence of the reductants supplied by the DHA to DHAP pathway.
The genes responsible for the conversion of glycerol to 1,3-propanediol have been isolated and are all encompassed by the dha regulon. In order to make use of the potential advantages of higher protein expression and growth rate of recombinant bacteria, several attempts have been made to express the dha regulon as heterologous genes in E. coli. For example, the dha regulon from Citrobacter (Daniel et al., FEMS Microbiol. Lett., 100, 281, (1992)) and Klebsiella (Tong et al., Appl. Environ. Microbiol., 57, 3541, (1991); have been expressed in E. coli and have been shown to convert glycerol to 1,3-propanediol. The expression of the dha regulon in recombinant bacteria offers potential advantages over wild type production of 1,3-propanediol. The genes involved in the dha regulon provide both the enzymes and the necessary reductants needed for the efficient conversion of glycerol to 1,3-propanediol. However, simultaneous overexpression of both glycerol dehydrogenase and glycerol dehydrates results in some of the glycerol being converted to DHA. It would be advantageous to convert all the glycerol to 1,3-propanediol by expressing only the reductive pathway enzymes while providing a different substrate for the generation of reductant. A preferred system would provide for a more efficient use of the glycerol substrate while maintaining high yields of diol product.
It has long been known that a number of bacteria are capable of using 1,2-propanediol is a sole carbon source. It is thought that this ability is conferred by a specific vitamin B.sub.12 dependent diol dehydratase which is encoded by the pdu operon. The pdu operon is linked to the cob operon which encodes enzymes needed for the biosynthesis of vitamin B.sub.12 and both operons are subject to the regulation of the same activator protein encoded by the c pocR gene.
Recently the genes encoding the diol dehydratase of Klebsiella oxytoca were cloned and sequenced and the genes were expressed in E. coli. Although active diol dehydratase was observed in these transformants, there is no evidence that these clones are able to metabolize a carbon substrate to 1,3-propanediol.
Various Salmonella and Klebsiella sp. are known to produce a diol dehydratase which catalyzes the conversion of 1,2-propanediol, under anaerobic conditions, to propionaldehyde and eventually to 1-propanol and propionic acid. The diol dehydratase has also been identified in Clostridia, and Propionibacterium but not in E. coli. The diol dehydratase from Klebsiella sp. can convert glycerol to 1,3-propanediol (Forage et al., Bacteriol, 149, 413 (1981)).
Although the primary function of the pdu diol dehydratase is in the metabolism of 1,2-propanediol, applicants have discovered that the expression of K. pneumoniae diol dehydratase in E. coli will catalyze the conversion of glycerol to 1,3-propanediol. The recombinant bacteria expressing the dial dehydratase pathway converts glycerol to the desired 1,3-propanediol product and is not dependent on a linked system as with the glycerol dehydratase system. Applicants have discovered that transformation of recombinant bacteria with the pdu diol dehydratase genes from Klebsiella sp. affords a new, efficient and cost effective biological route to 1,3-propanediol.